DE2539258A1 - IMMUNOLOGICAL METHOD OF ANALYSIS FOR THE DETERMINATION OF TOTAL THYROXINE IN BLOOD SERUM - Google Patents

Description

The determination of the total thyroxine contained in the blood serum (Τ.) Is used to support the diagnosis of thyroid function and a therapy based on it. The test is carried out in vitro, which avoids that the patient is exposed to radioactive rays. All you need is a blood sample from the patient. The with help T, analysis carried out on a radioactive isotope is permitted the assessment of thyroid function in certain circumstances where other standard methods such as determination of protein-bound iodine, because of the presence of various Forms of iodine are not applicable. The radioimmunological T, analysis methods can be used after administration of iodine-containing compounds and also in the course of drug treatment the thyroid. The determination of the serum total thyroxine opens up a way to measure the direct Thyroid hormone secretion (as thyroxine).

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T ^ analysis methods require a means of separating the free Hormone from the bound, the conventional practice for the extraction of the protein charcoal, double-antibody -Working methods or uses ethanol. However, these methods are complex, time-consuming and require a great deal of Accuracy.

The invention provides a simple, accurate and quickly practicable one radioimmunological analysis method for determination of serum total thyroxine, in which the protein with the help precipitated by trichloroacetic acid and treated with a base such as sodium hydroxide, is resolved. One can use the acid and base in separate stages or as a mixture instead of others Use materials such as ethanol for protein extraction. Since a 100 percent extraction is achieved, there is none Correction required.

The simplicity of the analysis method according to the invention lies in that no centrifugation, evaporation, transferring of samples or reagents, column preparation or weighing of reagents are necessary. Furthermore, all analysis steps can be carried out in the same tube. Since the inventive Analysis method none of the complex measures mentioned above requires, it can be carried out more quickly than the conventional methods. Furthermore, there is no need to collect the extract for its extraction to take place. The analysis method according to the invention has a high specificity to the extent that substances which affect competitive protein binding or other processes or have a corresponding ability to have no effect. Because a smaller number and smaller quantities of reagents are required, the analytical method is economical and can be routinely used in human and veterinary medicine use. Suitable because of the extremely small amounts of test sera that are required to carry out them the method is particularly suitable for paediatrics and research on small laboratory animals.

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To carry out the radio immunological according to the invention The substances and reagents required for the analytical procedure are prepared as follows:

T. Antibodies are generated in rabbits which are immunized against bovine Thyreoglobulin (1 percent emulsion in Freund's adjuvant) once a week for 3 weeks. Blood samples are generally taken from the test animals 8 weeks after the primary immunization. The strength of the antiserum is such that the antibodies bind about 60 % of the tracer dose of J- ¹ S, (corresponds to 2 / ug / 100 ml). The dilution of the antiserum (1:20) for the analysis is carried out as follows: 25 ml of antiserum are mixed with 15 ml of propylene glycol and 5 ml of 1% phenol and the resulting solution is made up to 500 ml with phosphate buffer (1 m; pH 7.4) on. Before dilution, the antiserum is labeled with JT (0.5 / Ug JT // 25 ml antiserum).

¹²⁵ JT
⁰ - ^L 4

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Commercially available thyroxine labeled J is used. The labeled substance has a specific activity of about 60 / uc // ug and is supplied as a 50 percent propylene glycol preparation delivered.

T, standards:

Use reagent grade thyroxine (free acid). After drying at 40 ° C. (overnight), appropriate proportions are obtained weighed in and dissolved in a very small amount of 0.1 η NaOH. The standard solutions are prepared in 0.25 percent albumin.

Thyronine-free serum :

The T «standards are made with thyronine-free human serum produced by adding 20 g of activated charcoal (charcoal) to 100 ml of collected normal human serum became. The slurry is 1 hour at room temperature

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slowly stirred, then 24 hours in a cold room (8.9 to 1O ⁰ C) allowed to stand, then centrifuged three to five times and finally the series by filtration subjected Millipore filter.

The radioimmunological analysis method according to the invention is carried out as follows:

Procedure ;

25 microliters of serum are pipetted into tubes and 0.1 ml of 30 percent trichloroacetic acid are added to precipitate the plasma proteins added. After shaking the tubes for 5 minutes, 0.3 ml of 0.75 η sodium hydroxide solution is added to the solution from the native T.-binding proteins to thyroxine released. The addition a trichloroacetic acid-NaOH mixture is also possible like the separate addition of the trichloroacetic acid and the sodium hydroxide solution and simplifies the process by a further step. To

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5 minutes of shaking the tubes are added 0.6 ml JT, -labeled thyroxine bind end it rabbit anti-bovine serum thyroglobulin (1: 20; pH 7 »4) and shaking the tube again for 5 minutes. The tubes are then brought to equilibrium for 15 minutes at room temperature and then placed in an ice bath (2 to 4 ° C.) for a further 15 minutes. Then insert an anion exchange resin sponge into each tube and compress it with the help of a plunger. The radioactivity (initial activity) is determined in a few tubes. After incubating with the reaction components for one hour, the sponges are washed four times with deionized water and the radioactivity (final activity) is determined. The radioactivity uptake of the sponges (final activity / initial activity χ 100, measure for the ³ JT, -. Displacement of antiserum) is linearly related to the thyroxine present in the standard or test serum (up to 20 / Ug T4 / 100 ml serum or more, depending on the type and degree of dilution of the T * -binding antisera).

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T. calibration line (standard curve), accuracy and sensitivity ;

T ,, calibration ;

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If you compare the percentage Y-T. Uptake of the sponges plots the T, component, there is a linear relationship. With suitable dilution of the antiserum and adjustment of the volume of the reaction components, one can even then achieve linear dependency if you use a test serum

125 volume of 0.1ml works. The linear relationship of the JT ^ - uptake of the sponges over the entire tested range of the T. standards (0 to 20 / ag T ./100 ml serum) indicates that the analytical sensitivity is uniform over the entire measuring range; on the other hand, the sensitivity increases at high T. values, which is expressed by a curve relationship

Analysis accuracy ;

The accuracy of the analytical procedure is determined in two ways;

Accuracy within the same analysis ; To determine the "internal" analysis accuracy. the coefficient of the variation between the repetitions of the test is calculated on three serum samples with high, medium and low T i concentration; the results can be seen from the comparison below;

Number of mean value standard fluctuation determinations (/ ug / 100 ml) deviation coefficient, ^

Serum with

low 20 T ₄ value

Serum with

middle, 20 normal
3? ₄ value

Serum with 18 high T. value

Analysis to Analysis Accuracy ;

To determine the accuracy from analysis to analysis, the coefficient of variation between determinations is calculated which

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5.0 0.29 5.8 7.5 0.72 ■ 9.6 14.6 0.80 5.5

Λ *

be performed in sequential analytical procedures using different lots of reagents (including fresh labeling of aliquots of the frozen immune sera). The results are from the following. Comparison visible:

Number of mean value standard fluctuation determinations (/ ug / 100 ml) deviation coefficient, $>

j ———————

collected

normal _1C - _ _RF . _ft

human ^{li? 7} ' ³ °> ^{ö6 11} > ⁸

sensitivity i

The lower limit of the sensitivity of the method is 0.8 / µg / 100 ml. To determine the sensitivity, the effect of constant increases of 0.2 / µg ¹ T, (/ 10OmI), starting from 0.2 / µg T, is tested , up to 1 / ug T, / 100 ml. The lowest T, concentration at which a change in the radioactivity taken up by the sponge can be determined is determined as the lower sensitivity limit of the analysis. While a higher sensitivity (i.e. change in the sponge uptake for a given increase in T.) can easily be achieved by diluting the antiserum, especially in the lower part of the calibration line (area of the hypothyroidism), there is no particular advantage in terms of separating the T. values to recognize different thyroid conditions. Furthermore, one finds a curve drawing with extreme sensitivity, ie at higher T 1 contents the differences in the reaction caused by the uptake of the sponge are not great.

Specificity ;

To assess the specificity of the analysis, T. values are determined on collected normal human serum before and after adding various substances. Types and amounts of substances tested can be seen from Table I. The table shows that none of the tested substances, with the exception of T, in high amounts (such proportions by far exceed even therapeutic

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table doses) somehow affects the analysis. Such a high degree of specificity appears particularly important when considering the test substances, some of which influence the T «values in conventional analytical methods based on competitive protein binding. The various antidiabetics, salicylates, anti-coagulants, organic and inorganic dyes and radio-opaque materials do not affect the analysis, nor do Dilantin in a dose of 500 / Ug / ml. Complement, which has been found to interfere with the double-antibody separation method has no effect on the analysis - presumably because of its denaturation in the presence of trichloroacetic acid / NaOH. The lack of effect of glucose shows that the method is not affected by Osmolalitätsänderun- gen. The lack of influence of immunoglobulins is particularly interesting, not only because of the lack of interference from antibodies (antithyroglobulin itself), but also because the analysis cannot easily be influenced by changes in the protein concentration.

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3159, _g .

Table I

Specificity of immunological thyroxine analysis

T the collected human per ml trains- ·

Serum added substances set amount (/ ug / 100 ml)

O 7.2 + 0.5

Arthropan ♦ - 500 / ug 7.7

Iodoacetamide. 100 / µg 6.8

Sodium iodide 100 / ug 6.3

Phenoformin HCl 50 mg 6.7

Tolinase 50 / ug 6.7 heparin 1000 units 6.8

Dilantin 500 / µg 7.9

3-iodine-1-tyrosine 50 / µg 7.2

Immune serum globulin (human) 50 / µg 7.3

Goat antiserum against human TG * 0.1 mg 7.3

Glucose 100 / ug 6.3

3,3,5-triiodo-i-thyronine 100 / µg > 80

Hypaque 6000 / µg 7.0

Phenylbutazone x 100 / µg 7.3

* TG = thyroglobulin

The method of the invention requires a means of separation

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of the unbound J-thyroxine from that bound to the thyroxm- binding globulin. Appropriate release agents are ion exchange resins. These can be used as granules. However, since centrifugation is necessary in the case of granules, it is preferred to use resin sponges which contain ion exchange resin particles distributed in a plastic sponge. In a preferred embodiment of the invention, the unbound I-thyroxine is separated from the bound thyroxine with the aid of an anion exchange resin sponge. The amount of thyroxine contained in the patient's serum can be determined by comparing the percentage uptake of the sponge with a standard curve.

The resin sponge used in the present invention consists of one

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Polyurethane foam with interconnected cells, which is a strongly basic anion exchange resin contains; See U.S. Patents 3,024,207 and 3,094,494. Such polyurethane foam resin sponges can thereby be produced v / ground that the ion exchange resin particles are a mixture of a polyether or polyester xind a polyisocyanate incorporated and subject the approach to the usual conditions for the production of polyurethane foams. An example for a suitable ion exchange resin is a strongly basic anion exchange resin in the chloride form, as described below called "Amberlite IRA400" is on the market. Such Resins can be prepared according to that described in U.S. Patent 2,591,573 Process are produced. The resin used should not remove any iron that is already bound to the globule molecule still bind the entire molecule.

The resin sponge can be produced in various shapes. A cylindrical plug or block is useful, which can be easily inserted into the bottom of a container which is inserted into the borehole of a conventional scintillation counter is insertable. The actual dimensions of the cylindrical resin sponge block will be determined by that used in the test Serum volume and determined by the size of the container used to hold the serum and the radioactive solution. It is expedient to use a resin block with standard dimensions and a standard serum volume in the method according to the invention to use. The type of resin, the resin content of the polyurethane foam or the composition and the properties of the sponge can be varied in the most varied of ways. Variations are also possible with regard to the serum volume and the amount of the tracer substance. The feasibility of the analytical process is not affected by such modifications. The greatest possible benefits can be obtained with the help achieve the method according to the invention by having a certain serum volume and certain dimensions of a spe-, selects a special resin sponge as the norms.

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The use of too high a concentration of trichloroacetic acid leads in the radioimmunological analysis method according to the invention to break down the protein. It is therefore preferable to work with a less than 40 percent trichloroacetic acid solution.

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Claims (1)

3rd September 1975 Claims before Γ 1 .yRadioimmunological analysis method to determine the Total thyroxine in a serum sample, thereby characterized by adding trichloroacetic acid and sodium hydroxide to the serum sample mixed, incorporating radioactively labeled thyroxine into the mixture, allowing the mixture to come to equilibrium, the mixture with a resin sponge made of a polyurethane foam with interconnected cells, which contains a strongly basic anion exchange resin, brings into contact, the mixture and the resin sponge incubated, the initial radioactivity of the combination of mixture and Resin sponge determined with the help of a suitable measuring device, removes the resin sponge from the mixture, washes out the resin sponge and
the residual radioactivity in the resin sponge is determined. 2. The method according to claim 1, characterized in that the 125 radiolabeled thyroxine labeled with iodine ^J. 3. The method according to claim 2, characterized in that the mixture and the resin sponge are incubated for about 1 hour. 4. The method according to claim 3, characterized in that the trichloroacetic acid has a concentration of less than $ 40. 5. The method according to claim 4, characterized in that one 25 microliters of the serum sample with 0.1 ml of 30 percent trichloroacetic acid solution and 0.3 ml of 0.75 η sodium hydroxide solution were added - 11 - 609812/0737